The Impact of Intracellular Environmental Factors on htt Aggregation

Breanna Hodges*, Justin Legleiter and Sharon Groover, C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26505

Field (Broad Category): Chemistry (Physical Sciences & Engineering) 

Student’s Major: Chemistry 

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder which causes chorea, psychiatric disorders, and disrupted cognitive thinking. A mutation in the htt gene results in expansion of a polyglutamine (polyQ) domain in the huntingtin protein (htt), with expansion beyond 35 repeats resulting in HD. Expansion directly promotes the formation of toxic htt aggregates. Aggregation is complex and heavily influenced by a variety of chemical factors within the intracellular space. These factors include the crowded cytosolic space and membranous surfaces associated with organelles; however, mechanistic details of how these specific features influence aggregation remain elusive. To determine how molecular crowding and the surface of the endoplasmic reticulum (ER) impact htt aggregation, a series of experiments using mutant htt proteins and peptides were performed. To mimic the crowded environment of the cytosol, htt aggregation reactions were analyzed in the presence and absence of chemically inert macromolecular crowders: dextran, ficoll, and polyethylene glycol (PEG). A fluorescent dye, thioflavin T (ThT), was used to monitor the kinetics of htt aggregation. Atomic force microscopy (AFM) was used to visualize aggregate morphology, and lipid binding assays were used to determine how a crowded environment alters htt’s surface activity. Dextran and ficoll enhanced aggregation; PEG reduced it. A similar pattern was observed in how crowders impacted htt’s surface activity. In addition, preliminary studies were performed to determine how the presence of ER purified from murine brains influences htt aggregation. Currently, the assays used are being optimized to compensate for noise associated with the addition of ER. 

Funding: 

Program/mechanism supporting research/creative efforts: WVU's Research Apprenticeship Program (RAP) & accompanying HONR 297-level course